The overall goal of this long-term project is to delineate the specific structural and functional interactions involved in prolactin receptor recognition and signal transduction. Although best known for its traditional role as a pituitary-derived hormone, recent research has established important autocrine/paracrine functions of prolactin in the growth and development of a diversity of tissues. Expression of prolactin and its cell-surface receptor has been demonstrated in multiple human breast and prostate cancer cell lines. Prolactin has mitogenic and angiogenic function in these tumors and increases cancer cell motility. The biology of peripheral prolactin synthesis is distinct from the pituitary, including alternative mechanisms for transcriptional regulation, RNA splicing, and hormone storage and secretion. Additionally, when isolated from peripheral tissue, or when produced by non-pituitary cell lines grown in culture, glycosylated and phosphorylated variants of prolactin are found. Research has demonstrated functional consequences of these modifications, some of which may act to counter the tumorigenic effects of native prolactin. Similarly, multiple isoforms of the prolactin receptor have been described in both healthy and malignant tissue. These receptor isoforms differ in their affinity for prolactin and in their activation of intracellular signal transduction pathways. The central hypothesis of this application states that distinct subsets of prolactin residues are responsible for the differences in recognition specificity displayed by prolactin and its variants towards prolactin receptor isoforms. We will use NMR spectroscopy to identify, at an atomic level, the precise intermolecular interactions responsible for receptor recognition specificity. The functional significance of these interactions will be investigated by measurement of receptor binding kinetics and assays of cellular activation and signal transduction. By comparison of the wild type and variant forms of prolactin, we hope to uncover the molecular mechanisms for both receptor agonism and antagonism. The results of the proposed research will aid the development of potential therapeutic agents designed to modulate specific activities of prolactin, including prolactin receptor antagonists, which will have significance for the treatment of breast, prostate and, potentially, other cancers.